Advancing eDNA methods for monitoring the reproduction of quagga and zebra mussels in lakes

Abstract This study demonstrates the applicability of environmental DNA (eDNA) methods to monitor the reproductive periods of two invasive freshwater mussels, Dreissena polymorpha ( zebra mussel) and Dreissena rostriformis bugensis ( quagga mussel), addressing the need for improved detection and monitoring techniques for these ecosystem-altering species. New primers and probes for multiplex droplet digital PCR (ddPCR) were developed to enable the discrimination between zebra and quagga mussels, whose veliger larvae are morphologically indistinguishable. Three eDNA sampling methods were compared: integrated water samples (0-50 m depth), sub-surface water samples, and plankton bulk samples. These methods were applied in four deep peri-alpine lakes and the results obtained with eDNA were compared to traditional veliger microscopic counts. The values obtained with the three eDNA approaches are positively corelated with visual counting of veliger larvae, but integrated water and plankton bulk eDNA samplings were found to be more effective in quantifying dreissenid veliger larvae and estimating reproductive periods than sub-surface water eDNA. The bulk-based approach is robust for qualitative presence/absence veliger larvae information, with a positive eDNA signal only when larvae are observed; it is the only eDNA method that successfully detected zebra signals in Lake Geneva. However, an overestimation of winter reproduction signal was observed for one lake with this method. The water-integrated approach captures well the quantitative dynamics of counted veliger larvae, but with the presence of false positives reproductive signal (mussel eDNA signal when no larvae are observed). The eDNA approaches tested here have enabled the characterisation of the reproductive dynamics of each of the two species in the studied lakes, highlighting the fact that quagga can reproduce throughout the year in peri-alpine lakes where it is dominant, whereas in lakes dominated by zebra, the reproduction period is limited to the warm season. The study also provides the first clue of quagga mussel presence in Lakes Annecy and Aiguebelette. Although further methodological refinements are suggested for the eDNA approaches evaluated in this study, those approaches are nonetheless already applicable and can contribute to a better understanding of these invasive species ecology in lakes.